Patients with lung diseases are commonly treated with high concentrations of oxygen. Supplemental oxygen, while life sustaining, can cause lung injury. The specific cellular, subcellular, and molecular targets of hyperoxic lung injury have not been elucidated, hampering the rational design of specific interventions. Recent studies in premature infants, a population particularly predisposed to hyperoxic lung injury, and in mice exposed to hyperoxia found decreased expression of Clara cell secretory protein (CCSP) and increased reactivity of CCSP with 2,4-dinitrophenylhydrazine (DNPH), indicating oxidation of CCSP. These data suggest that Clara cell function and CCSP expression are adversely affected in hyperoxia. The working hypotheses of this proposal are that maintaining CCSP expression and Clara cell function in hyperoxic animals is protective against hyperoxic lung inflammation and injury and that products of oxidation of CCSP in hyperoxic animals in vivo will be characteristic of a limited number of biologically feasible mechanisms of oxidation studied in vitro. Specific Aim 1 is to test the hypothesis that intratracheal administration of recombinant CCSP will maintain CCSP expression in mice in the course of exposure to hyperoxia and will be associated with attenuation of hyperoxic lung injury and inflammation. This is based on the evidence that enhanced glutathione reductase (GR) or manganese superoxide dismutase activity in mitochondria protects cells from oxidant stresses, including hyperoxia. Specific Aim 2 is to test the hypothesis that enhanced expression of GR in the mitochondria of Clara cells in mice will prevent the hyperoxia-induced decrease in CCSP expression in association with increased resistance to hyperoxic lung injury and inflammation. We will enhance Clara cell expression of GR in the mitochondria and cytosol. We also will enhance expression of MnSOD in Clara cells. In these three separate transgenic lines of mice and wild-type mice, we will determine the effects of the enhancements on responses to hyperoxia. Finally, Specific Aim 3 is to test the hypothesis that the specific molecular modifications of CCSP in lung epithelial lining fluid of animals exposed to hyperoxia will be characteristic of the specific mechanisms responsible for oxidation of CCSP, which, in turn, are responsible for loss of immunoreactivity and loss of function of CCSP. The results of the studies described in this proposal are designed to provide the foundation for the rational design of therapeutic interventions in humans.